Although p53 has long been known as a tumor suppressor, whether its recently discovered family member, p73, is also a tumor suppressor gene remains controversial. Data from some human tumors, including lung adenocarcinoma, transitional cell carcinoma of the bladder, osteosarcoma, mammary adenocarcinoma and myelogenous leukemia, indicate that loss of p73 correlates with tumor formation while other data has shown that it is overexpressed and thus behaves as an oncogene. Much of the controversy regarding the role of p73 is due to the existence of numerous p73 isoforms with potentially opposing functions and the lack of antibodies to distinguish between them. p73-null mice have been generated, but most of these mice die at approximately 3 weeks of age, which precludes tumor analysis. We have shown previously that p73-/- mice that live to adulthood develop lung adenocarcinomas at high frequency (60%). Interestingly, p53+/-;p73+/- mice develop a complex tumor spectrum including lung adenocarcinoma, many of which are metastatic. This points to a role for p73 in tumor suppression and metastasis. We wish to determine how p73 and its isoforms may regulate cellular processes that lead to tumorigenesis and metastasis. Study of p73 is challenging, though, because of the existence of multiple isoforms of this gene. These isoforms fall into two main categories-the transactivation (TA) isoforms, which have a transactivation domain, and the ?N isoforms, which lack the transactivation domain. The role of these isoforms in cancer is unclear, and data from human tumors are correlative. Data from other labs have shown that the TA isoforms have activities more like those of p53 whereas the ?N isoforms act as dominant-negatives against p53, TAp63, and TAp73. Interestingly, some recent reports indicate that ?Np73 itself can transactivate target genes and that expression of this isoform in cells at physiologic levels does not lead to increased cellular growth. This indicates that the ?N isoform may have tumor suppressing in addition to promoting activities. We hypothesize that TAp73 suppresses tumorigenesis and metastasis whereas ?Np73 may suppress or promote tumorigenesis depending on cellular context. The Specific Aims in this application are designed to elucidate the roles of p73 and its isoforms in tumor suppression and metastasis using genetically engineered mice carrying conditional knockout alleles of either the TA or ?N isoforms of p73. The existing p73-/- mouse models are not suitable for our research because these mice lack all isoforms of p73, making it impossible for us to determine the genetic contribution of each isoform to tumorigenesis and metastasis, and because most p73-/- mice die early in life (~ 3 weeks of age), making it difficult to understand the consequences of p73 loss in adult mice. We plan to generate new mouse models to overcome these limitations. The Specific Aims of this proposal are: 1. To determine which isoform(s) of p73 (TAp73 and/or ?Np73) play a role in tumor suppression and metastasis by characterizing primary mouse cells expressing each of these isoforms individually. 2. To determine which isoform(s) of p73 (TAp73 and/or ?Np73) play a role in tumor suppression and metastasis by characterizing in vivo mouse models of tumorigenesis and human patient tumor samples. 3. To identify genes that are direct transcriptional targets of p73 and its isoforms in the process of tumorigenesis, tumor progression and metastasis using a mouse model of lung adenocarcinoma and human patient tumor samples. The generation of these isoform specific p73 knockout mice and the analyses of mouse and human lung tumors will clearly define the mechanisms employed by TA and ?N p73 in tumorigenesis and metastasis. Data from this proposal will aid in novel therapies for cancer patients with mutations in the p53/p73 pathway. PUBLIC HEALTH RELEVANCE: p53 is frequently mutated in human tumors. The role of the p53 family member, p73, in tumorigenesis is poorly understood. The function of p73 in tumorigenesis has been difficult to determine due to the existence of multiple isoforms with apparently opposing functions. The goals of this grant proposal are to use genetically engineered mice generated in my laboratory that express these isoforms individually to determine their function in tumorigenesis and metastasis. A genome wide approach will be used to identify direct targets of p73 and its isoforms in tumorigenesis and metastasis. Additionally, the status of the p73 isoforms and targets will be ascertained in human lung tumors and will be correlated with p53 status and response to chemotherapy. Lastly, the function of p73 targets in metastasis will be determined using in vitro and in vivo techniques. These studies will unveil the functions of the p73 isoforms in tumorigenesis and metastasis and have important clinical implication for patients with mutations in the p53/p73 pathway.